JPH11507402A - Molecularly imprinted granular polymer and its stabilized suspension polymerization in perfluorocarbon liquid - Google Patents

Abstract

  (57) [Summary] As a disperse phase, it does not interfere with the interaction between the functional monomer and the print molecule required for the recognition process during molecular imprinting and has a particle size in the range of less than about 5 μm to about 100 μm The present invention provides a suspension polymerization method for molecular imprinting, which is based on using a perfluorocarbon liquid and can be prepared by the following method.

Description

DETAILED DESCRIPTION OF THE INVENTION          Molecularly imprinted granular polymers and their polymers              Stabilized suspension polymerization in perfluorocarbon liquidField of the invention   The present invention relates to a molecularly imprinted polymer support. The present invention relates to a carrier and a method for producing these supports. More specifically, the present invention provides the following formula: Using a suspension polymerization method using a stabilized copolymer having olecule) around the polymerizable functional monomer. Where:   X is C_nF_{2n + 1}, C_nF_{2n + 1}(CH_Two)_rO-, C_nF_{2n + 1}O- or C_nF_{2n + 1}SO_Two N (C_TwoH_Five) C_TwoH_Two-O;   Y is (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two-O or (Z)_tCH_ThreeO (CH_TwoCH_Two O)_m-Where Z is a printed molecule;   n is from 1 to 20;   m may be from 0 to about 500;   p is at least 1;   q may be 0 or a positive number;   r is 1-20, preferably 1 or 2;   s is 0 or 1; and   t is 0 or 1. The polymeric support of the present invention is preferably in a granular form, which comprises amino acids, amino acid derivatives. Enables the separation or resolution of the body, pharmaceutical compounds and polysaccharides.Background of the Invention   Molecular imprinting, also called templating, Is used for chiral separation, which includes a polymerizable functionality around the print molecule. And arranging the functional monomers. This is the print molecule and the functional monomer When Using non-covalent interactions such as hydrogen bonding, ion pair interactions, etc. (Non-shared imprint) or by its reversible covalent interaction (shared imprint) Print). The resulting complex is further combined by polymerization to form a highly crosslinked porous It is a polymer matrix. When the printed molecules are extracted, special A site with an irregular shape and functional groups corresponding to the original print molecule remain. Mosbach, K., Tr ends, Biochemical Sciences,7, 92-96 (1994); Wulff, G., Trends, Biote. chnology,1185-87 (1993); and Andersson et al., Bioseparations (Ngo. Ed.), Pp. 383-394, Molecular Interactions.   Various racemates are resolved by molecular imprinting. And ersson et al., Bioseparations (Ngo.TT), Plenum Press, pp. 383-394 (19). 93) "Amino acid derivatives" in Molecular Interactions; . Am. Chem. Soc.,113, 9358-9360 (1991); "Drugs"; Kempe et al. Chromatogr.,664, 276-279 (1994); Wulff et al., J. Org. Chem.,56, 395-400 (1991) "Sugars"; and Mayes et al., Anal. Biochem.,222, 483-488 (199 See 4). In many cases, Baseline resolution is performed.   In contrast to other chiral stationary phases, the benefits of molecularly imprinted polymers The point is that the elution order of the enantiomers is predictable. Imprintable support The support is manufactured by a bulk polymerization method, in which a block of porous polymer is used. For the production, a porogenic solvent is used. But bulk The polymeric support is crushed and ground when used in separation columns and analytical protocols, It must be sieved to an appropriate particle size. For example, chromatography In the case of the measurement using a polymer, polymer particles having a size of usually less than 25 μm are used. But bal In order to supply such small particles in the polymerization method, a pulverization method must be used. , This is not satisfactory. Grinding methods involve irregularly shaped particles and excess Undesirable amounts of "fines" are obtained. Usable among normally ground polymers Less than 50 percent (50%) is recovered as usable particles. Generally irregular Column cannot be efficiently packed with small particles and scale-up It has been found that difficulties are often encountered during the backup stage. But well-formed grains Particles, such as particulate polymers, may be preferred in most cases. Also powder Crushing method Requires additional processing steps to remove fines, i.e., settling steps. I do. This is expensive and time consuming. Bulk polymerization and the necessary grinding The law makes the effort of this conventional law severe, wasteful and unacceptable.   Manufacture beads from acrylic monomers that can contain imprinted molecules Therefore, attempts have been made to use suspension and dispersion polymerization methods. In theory, this These suspension and dispersion polymerization methods could be an alternative to bulk polymerization. You. However, existing suspension and dispersion methods use relatively hydrophobic monomers. Water or a highly polar organic solvent (eg, alcohol) as the continuous phase It is not satisfactory because of its use. These solvents are Has a specific interaction with the solvent, and is likely to compete with the functional monomer for the solvent. It is incompatible with most covalent and noncovalent imprint mixtures. In the suspension polymerization method , Because the solvent is used in a considerable molar excess, the solvent saturates the monomer phase and Significantly reduce the number and strength of interactions between the reactive monomers and the print molecules. Further In addition, acidic monomers are highly soluble in water, and may No coalescence or crosslinking occurs. Water-soluble print molecules are distributed in the aqueous phase. It is lost to spill. Not unexpected, but suspension polymerization in water Attempts to produce molecularly imprinted polymer beads Attempts have yielded very poor results. Damen et al., J. Am. Chem. Soc.,102, 3265-3267 (1980); Braun et al., Chemiker-Zeitung,108, 255-257 (1984) There is a stable covalent or metal chelate bond between the functional monomer and the print molecule. In some cases, use in aqueous conditions will be possible.   Also imprinted in the pore network of the finished granular silica To produce composite beaded particles Attempts have been made. Norrlow et al., J. Chromatogr.,299, 29-41 (1984); Wul. ff, Reactive Polymers,Three, 261-2757 (1985) or TRIM. But This method requires careful operation and requires additional The amount of imprinted polymer is necessarily reduced by the beads themselves.   Sellergren, B., J. Chromatogr.,673, 133-141 (1994) and Sellergren, B., Anal. Chem.,66, 1578-1582 (1994) states that polar solvents are mixed in molecular imprints. The use of dispersion polymerization in compounds has been reported. This method uses a regular bead A random sediment is obtained rather than a sediment. Evaluable results are highly This is probably due to the fact that charged molecules were obtained. Probably due to competing solvent effects.   Therefore, it is simple, reproducible and without compromising the quality of the resulting imprint Granular poly, including molecular imprints that do not require additional grinding and sieving equipment There is a need for Ma's recipe. The molecularly imprinted polymer is uniform Something is also required.Summary of the Invention   The present invention relates to a molecularly imprinted polymer support, and to that by suspension polymerization. Related to their manufacture. The suspension method according to the present invention employs polyoxyethylene Molecular imprinting method using perfluorocarbon liquid containing steal group I will provide a. Compounds containing perfluorocarbon-polyoxyethylene esters Compounds are required for the recognition process during molecular imprinting. It does not interfere with the interaction between the functional monomer and the print molecule. "Support" grains By changing the amount of the stabilizing polymer or the method of stirring, the size of the  Adjustment is possible from μm to about 100 μm.   It is therefore an object of the present invention to use imprinted polymers in granular and It is an object of the present invention to provide a method which can be easily produced in almost quantitative yield of possible substances.   Another object of the present invention is to provide a functional monomer and a printed component during suspension polymerization. Fluorocarbons that stabilize emulsions without disturbing interaction with the To provide a copolymer.   Further objectives include functional monomers, crosslinkers, print molecules, initiators and porologies. To stabilize the emulsion of the enic solvent.   Another object of the present invention is to provide a molecularly-sized particle having a size of about 2 μm to about 100 μm. It is to provide printed polymer beads in high yield.   It is a further object of the present invention to provide an excellent separation at low back pressure, high speed diffusion and higher flow rates. It is to provide a small bead packing of about 2-5 μm, which results in separation.   These and other objects and advantages will be more fully understood from the following description and accompanying figures. Will be clear.Brief description of figures   FIG. 1 shows 1.84 g of EDMA, 0.16 g of MAA, 4.2 g of copper in 20 ml of PMC. Addition for emulsification of roloform and 20 mg of AIBN for "standard polymerization" The graph shows the diameter of the beads against the obtained amount of PFPS.   Figures 2a-2e show beads produced by suspension polymerization in PMC according to the invention. 3 is a scanning electron micrograph of the sample. Here the beads are placed on aluminum pegs And use a Polaron E5150 coater to spatter it with 15 mm gold. I did it. Images were taken at 25 kV using an ISI 100A SEM. Magnification is 500x It is. 2 (a) PF2; 2 (b) PF10; 2 (c) PF13; 2 (d) PF14; 2 (e) PF15.   FIG. 3 shows the H level when Boc-D and L-Phe were separated on a 25 cm column of Example PFII. It shows traces of PLC. Conditions: Column is chloroform (CHCl)_Three) + Equilibrated with 0.1% acetic acid (AA); injected 20 μg Boc-D, L-Phe Elution was performed using a medium at a flow rate of 0.5 ml / min. Chromatograms were recorded at 254 nm.   4a-4e show a 25 cm column of Example PF15 (5 μm TRIM beads). It shows HPLC traces when Boc-D and L-Phe were separated. Condition: mosquito The rum was equilibrated with chloroform + 0.25% acetic acid; 1 mg in 20 μl mobile phase Of Boc-D, L-Phe, and using the same solvent, the flow rate was 4 (a) 0.5 ml / min; 4 (b) 1 ml 4 (c) 2 ml / min; 4 (d) 3 ml / min; 4 (e) 5 ml / min. At 0.5 ml / min, With f / g = 0.89, Rs = 1.36 and α = 1.52, the f / g values are 0.5, 1, 2, 3, and 5 ml / At the minute, they were 0.89, 0.89, 0.85, 0.76 and 0.61, respectively.Detailed description of the invention   According to the present invention, a liquid perfluorocarbon containing a polyoxyethylene ester group Based on an emulsion of a non-shared imprint mixture formed in the bon A suspension polymerization method is provided. Most of the conventional suspension and dispersion methods are relatively sparse. Water or a highly polar organic solvent (e.g., alcohol) as the mobile phase for aqueous monomers Call), which is not satisfactory in this regard. Therefore, another An approach is needed. The present invention provides this "alternative approach". It is. In addition, there are problems with conventional methods, such as specific interactions with printed molecules. Mostly shared and non-shared due to competition between solvent and functional monomer Solvents that are incompatible with the imprint mixture are avoided. In the present invention, print Use dispersants to prevent the interaction required for recognition between the molecule and the functional monomer. You do not need to know. In the present invention, a monomer, a crosslinking agent, a print molecule, a porogenic solution Liquid particles containing a solvent and a fluorinated surfactant. perfluorocarbons including polyoxyethylene ester groups Use a polymer. At this time, the number of print molecules for imprinting the surface is one. Sometimes it's a cord, sometimes it's not.   The stabilizer / dispersant of the present invention generally has the formula: Where:   X is C_nF_{2n + 1}, C_nF_{2n + 1}(CH_Two)_rO-, C_nF_{2n + 1}O- or C_nF_{2n + 1}SO_Two N (C_TwoH_Five) C_TwoH_Two-O;   Y is (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two-O or (Z)_tCH_ThreeO (CH_TwoCH_Two O)_m-Where Z is a printed molecule;   n is from 1 to 20;   m may be from 0 to about 500;   p is at least 1;   q may be 0 or a positive number;   r is 1-20, preferably 1 or 2;   s is 0 or 1; and   t is 0 or 1. C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Two-OH and (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two Blocks, triblocks and multiblocks formed from -OH monomers Copolymers are also within the scope of the present invention. A preferred stabilizer / dispersant of the present invention has the formula:             C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_TwoO-CO-CH = CH_Two And a monomer A defined by the formula:             CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_TwoO-CO-CH = CH_Two Wherein n is from 1 to 20 and m is 0 to 500. Preferably, n is 7.5 and m is about 43. Most preferred A new stabilizer / dispersant has the formula: This means that spherical beads can be produced in high yield by the suspension polymerization method. it can. Where:   X is C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Two-O;   Y is Z_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_TwoAt -O, Z is a printed molecule;   n is from 1 to 20;   m may be from 0 to about 500;   p is at least 1;   q may be 0 or a positive number;   s is 0 or 1; and   t is 0 or 1. Preferably, m = about 7.5, n = about 43, p = about 19, and q = about 1. m, n, The values of p and q need not necessarily be integers.   By using the stabilizer of the present invention, the printing molecule and the functional monomer Using non-covalent interactions such as hydrogen bonding, ion-pair interactions, etc. Imprint) or by reversible covalent interaction (shared imprint) The purpose of the physical properties of the dose is achieved. The formed complex is further added by polymerization. Highly crosslinked porous polymer obtained from copolymerization of different acrylic monomers Mermatrix. When the printed molecules are extracted, special The site of the shape and the functional groups corresponding to the original print molecule remain.   Print molecules that can be used in the present invention include, but are not limited to: It is not something to be done.   1. D- and L-Boc tryptophan   2. D- and L-Boc phenylalanine   3. D- and L-phenylalanine   4. D- and L-Boc-proline-N-hydroxysuccinimide ester   5. D- and L-Cbz-tryptophan   6. D- and L-Cbz-aspartic acid   7. D- and L-Cbz-glutamic acid   8. D- and L-Cbz-tryptophan methyl ester   9. Nitrophenyl α and β, D-, L-galactoside   Ten. (S)-(-)-Timolol   11． D-fructose   12． D-galactose   13. Phenyl α-D-mannopyranoside   14. Acrylic α and β-glucoside   15． (R) -phenylsuccinic acid   16． Ac-L-Trp-OEz   17． L-PheβNA   18． L-LeuAn   19. L-PheAn   20. L-PheGlyAn   twenty one. L-MenPheAn   twenty two. L-PyMePheAn   twenty three. L-PLPheAn   twenty four. N-Ac-L-Phe-L-Trp-OMe   twenty five. Diazepam   26. Propranolol   27. ephedrine   However, in the following non-limiting examples, Boc-D- and L-Phe, and L-Phe were used. Was.Preparation of reagents monomer   Ethylene glycol dimethacrylate (EDMA) and methacrylic acid (MAA) (Merck, Darnstadt, Germany) was vacuum distilled before use. Trimethacrylic acid Limethylolpropane (TRIM) (Aldrich Chemie, Steinheim, Germany) , Styrene (Aldrich), methyl methacrylate (MMA) (Aldrich) and meth Benzyl acrylate (BMA) (Polysciences, Warringt, Mass.) on) was used as is. 2,2'-azobis (2-methylpropionitrile) (AI BN) was obtained from Janssen Chimica, Goel, Belgium.   2- (N-ethylperfluoroalkylsulfonamide) ethanol (PFA-1 ) (Fluorochem, Old Glossop, UK), PEG2000 monomethyl ether (MME) (Fluka Chemical AG, Buchs, Switzerland) and PEG 350 MME ( (Sigma, St. Louis, Missouri) is a trademark of Acryloyl Chloride in dichloromethane. And acrylates by reaction with triethylamine. Purchase is also possible (from Fluorochem and Polysciences, respectively). Perful Oro (methylcyclohexane) (PMC) and Fluorad FC430 are also Obtained from Fluorochem.Imprint molecule   Boc-D-Phe, Boc-L-Phe and Boc-D, L-Phe were obtained from Bachem AG ( (Boc = t-butoxycarbonyl; Phe = F) from Bubendorf, Switzerland. Enylalanine).Porogenic solvent   Chloroform (CHCl_Three) (For HPLC) Ethanol is removed through a basic alumina column to use as a solvent Then, molecular sieves were put and stored. Used directly for HPLC. G Ruene was sodium and acetone was dried over molecular sieves before use. other The solvent of the grade for analysis or higher was used as it was.Preparation of stabilizer   The emulsion stable / dispersed polymers (PFPS) according to the invention are mostly Which formula: Where:   X is C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Two-O;   Y is Z_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_TwoAt -O, Z is a printed molecule;   n is from 1 to 20;   m may be from 0 to about 500;   p is at least 1;   q may be 0 or a positive number; and   t is 0 or 1; In particular, n is about 7.5, m is about 43, p is about 19, and q is about 1.Example 1   4 g (7.2 mmol) of acryloyl PFA-1             C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Two-CO-CH = CH_Two And 0.76 g (0.36 mmol) of the formula:             CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two-CO-CH = CH_Two Dissolve acryloyl PEG2000 MME as defined in 10 ml of chloroform Was. In the above formula, n is about 7.5 and m is about 43. 24 mg (76 μmol) of AIBN And dissolved oxygen was removed by flowing nitrogen for 5 minutes. And this tube Was sealed and the contents polymerized in a shaking water bath at 60 ° C. for 48 hours. The resulting solution Was slightly turbid, but became more turbid when cooled. Solvent slowly at 30 ° C under reduced pressure It is almost distilled off at 60 ° C under reduced pressure to avoid foaming. Removed. The resulting polymer is a sticky, pale yellow paste, which is It was used without any further treatment.   Other polymeric stabilizers use monomers and 1 mole% ATRN in appropriate ratios, Synthesized in a similar manner. All is obtained from cream as an amber polymer, which They varied from glassy to soft paste depending on their composition.Suspension polymerization   Determine the amount of porogenic solvent needed to just saturate 20 ml of PMC. Specified. In a 50 ml borosilicate glass tube, add the required amount of PFPS to the same amount of Porogeni. Solution, and add 20 ml of PMC to it to make a milky emulsion. , Shake until uniform white. 5 ml of “imprint mixture” (Table 1) In addition, the mixture was emulsified while stirring at 2000 rpm for 5 minutes.   * Beads made according to the present invention are listed in the first column of the table under the PF code number. This is the same in the comparative example.   The polymerization equipment used in all polymerizations of the present invention contained 50 ml of borosilicate glass. This tube has a screw cap and a stainless steel tube in the center. A hole has been drilled through the rotating shaft of a stainless steel flat blade stirrer. You. This flat blade stirrer is about 1.5 times the length of the tube. Evaporation through this hole A rubber seal was used to control. There is another small hole in the lid It can be used to send a stream of nitrogen into the tube. Stand retort tube Stand vertically in a (retort stand) and stir with an overhead stirrer. About 5 from tube  Place the UV lamp at a distance of cm and combine it with the lamp to maximize reflected light. The tube was wrapped in aluminum foil.   Prepare an emulsion of PF1-PF16 in Table 1 above and dispense with nitrogen for 5 minutes. The emulsion is then placed in a cooled nitrogen atmosphere at room temperature. While stirring at a flow rate of 500 rpm, UV of 366 nm was irradiated to perform polymerization. Polymerization is 3 Continued for hours. The obtained polymer particles (beads) are filtered with a glass filter, and PMC Was recovered. Wash the beads thoroughly with acetone to remove loose particles After sonication and crushing (large lumps are gently crushed and crushed with a spatula) It was dried and stored.   Adjust the size of beads by changing the amount of PFPS used during polymerization FIG. 1 shows this relationship. FIG. 1 illustrates the use of different amounts of PFPS in accordance with the present invention. 5 is a graph of the average value and the standard deviation of the beads manufactured as described above. Standard is 5 ml total volume Prepared using 2 g of monomer in it (see Table 1). Form a stable emulsion The minimum amount of PFPS possible is 10 mg, but this sample is also very agglomerated. I was in it. When PFPS is used at 150 mg or more, extremely small and irregular particles of 1-2 μm Only a child was obtained. No beads were found in these samples.Comparative example   Two commercially available fluorinated surfactants (polyfluoroalcohol (PFA-1) and Luorad 430), homopolymer of acryloyl PFA-1, acryloyl PFA-1 Series of random copolymers of styrene and styrene, methyl methacrylate or methacryl Benzyl acrylate and acrylates with perfluoroester and PEG groups Graft copolymers containing the main chain were also evaluated as stabilizers. However, this Most of them turned out to be ineffective. Emulsions quickly “creamy The reaction is hindered by the high density of dispersant generated, The dispersed droplets coalesced.   When polyacryloyl PFA-1 was evaluated alone, it was found that To give emulsions with sufficient stability to give good quality beads. Unfortunately Of the beads formed on the extremely hydrophobic surface after polymerization due to their low solubility. It was very difficult to remove this surfactant from the surface.   Copolymer of acryloyl-PFA-1 and acryloyl-PEG2000MME ( The most effective emulsions can be obtained using a molar ratio of 20: 1 with the latter being PFPS). Was.Properties of molecularly imprinted beads size   The type of emulsification depends on the polymerization method and the resulting molecularly imprinted beads. Affect. A preferred method involves stirring at about 2000 rpms for about 5 minutes. With this method, beads with a size of 2 to 25 μm have excellent uniformity. And reproducibility. Five separate polymerizations using 25 mg of PFPS as emulsifier The average bead size was 19.7 μm and the standard error was 0.6 μm.   Emulsification in an ultrasonic bath for 5 minutes, including significant amounts of small particles, Distribution was very extensive. If you have larger beads, i.e. 40 μm If you want 100 μm beads, shake well 3-4 times normally in a tube. The fruit was obtained.   The polymerization temperature also affected the polymerization method and the resulting beads. ABDV as initiator Tried to start with heat at 45 ° C, but only small and irregular pieces were obtained. Was done. Initiating polymerization by UV at 4 ° C resulted in very large masses wait. Due to the proximity to the UV lamp, the temperature increased somewhat during the polymerization, Most polymerizations were performed at ambient temperature (about 20 ° C), ie, above about 18 ° C. very In a warm climate, that is, when the ambient temperature reaches 30 ° C, Small beads are obtained, which gives reproducible results Suggests that the polymerization temperature may also affect the size of the beads.   Further, the polymerization is carried out in a range of solvents commonly used in molecular imprinting, for example, Runs were also made in lume, toluene, acetonitrile and acetone. In this polymerization method, All of these solvents can be used and are therefore almost always used for imprinting. Therefore, this law is appropriate. However, preferred solvents are chloroform and toluene. And acetone. The size and surface structure of the obtained beads are It also depends on the nick solvent. In a "standard" polymerization, 25 mg PFPS was used. If toluene (26 μm ± 12 μm-mean ± SD) is also acetone (52 μm ± 15 μm) Also gave larger beads than chloroform (18 pm ± 8 pm).Bead size distribution   The beads were suspended in acetone, dried on a microscope slide, and Approximately 150 beads were measured randomly using the reference line of the microscope. The magnification is Either 100 × or 400 × was used depending on the particle size. Some sumps Le also filmed with SEM. The measurements obtained from these images are optical measurements Well agreed with the results obtained fromSEM   Place polymer beads on aluminum pegs, Polaron E5150 Gold Sputter coating was performed with 15 nm gold using a coater. Furthermore, under different conditions To compare the size, surface and pore structure of the manufactured beads, ISI 100A Images were obtained at 25 kV using SEM. Scanning electrons of some granular polymer products Micrographs are shown in FIGS. 2a-2e. The method of the present invention can be used for various porogenic solutions. By using a medium, substantially spherical beads, that is, EDMA (2a-2 d) and give a polymer based on TRIM (2e). Surface depression The incidence of defects such as small holes is due to water-based suspension polymerization in water. Somewhat higher than the incidence normally observed in The form of the beads is suitable for suspension polymerization. Thus, as symbolized by the beads made, they are slightly denser and more slippery. The kana surface covers the more porous structure inside.   Beads made using acetone as a porogenic solvent (FIG. 2d) Was different. They are manufactured using chloroform or toluene Larger and the surface morphology is very rough, and moreover, There were many "debris". Using toluene as a porogenic solvent The beads produced have a lower surface shell density than those produced with chloroform. The internal structure was somewhat porous. FIG. 2b shows beads of polymer PF9 However, this has a lower percentage of crosslinking than the beads (polymer PF2) of FIG. 2a. The beads in FIG. 2b are much more irregular and distorted, indicating that their particles Are still much softer and more prone to deformation, so It suggests that it tends to be distorted. The internal form of these beads is a polymer It is also clear that it is more open and porous than PF2 beads. this This may contribute to the latter higher HPLC performance. FIG. The beads in (c) are PF13 beads, and the beads in FIG. 2 (e) are PF15 beads. Is shown.HPLC   The polymer beads produced according to the invention are molecularly imprinted And the properties of the recognition sites are at least as high as those obtained by conventional bulk polymerization. Imprint using Boc-Phe to confirm that it is as good as A series of polymers were evaluated by HPLC. This system is available at Boc-Phe There is a great deal of information on the properties of traditionally milled bulk polymers that have been imprinted. It was chosen because it can be obtained.   Suspend beads in chloroform-acetone mixture (17: 3) by sonication And slurries the slurry into 10 cm x 0.46 cm or 25 cm x 0.46 cm stainless steel. In a column, use an air driven fluid pump to remove the solvent. Filled at 300 bar as seton. Use 250 ml of methanol: vinegar Wash with acid (9: 1) and wash with chloroform containing 0.1% or 0.25% acetic acid. I was in equilibrium. 10 μg of Boc-D or L-Phe in 20 μl of solvent, or 20 μl g of the racemate and record the chromatogram at 254 nm at a flow rate of 0.5 ml / min. Was. Also, some separations use higher flow rates and higher loading compound loading Stay went. Chromatographic parameters were calculated using standard theory.   The results for the HPLC evaluation of the six polymers are summarized in Table 2. As the ratio of MAA to print molecule increases, non-specific interactions increase. Retention time is increased, which further increases the capacity factor. ors) increase. However, the α value is almost constant at about 1.8. This value is The values obtained under the same conditions for the crushed and sieved bulk Boc-Phe polymer (1.7 (Ranging from 7 to 2.17). The separation was performed with a MAA: Boc-Phe ratio of 12: 1. It was found to be 0.83 for the 10 cm column. Polymer -With longer columns (25 cm) of PF11, the The results were close to those of the enantiomeric Vaseline resolution reported in US Pat. This minute The chromatogram of the separation is shown in FIG. Polymer PF9 with only 62.5% cross-linking The ratio of lint molecules: MAA is the same, but the cross-linking is greater than 75% polymer PF11. Got good results. Previously, an increase in the degree of cross-linking within this range has been shown to improve separation. However, this was not observed in these examples. these It is impossible to change the parameters independently, increasing the number of binding sites Does separation improve even if the morphology of the polymer is changed by reducing cross-linking? Since it is unknown, PF11 is manufactured by reducing the amount of print molecules used. (See Table 1). From these observations, it can be seen that the improvement in separation depends on many compositions and It can be seen that this can be achieved by carefully optimizing the working variables. It can be even better if the bead polymerization method is simplified and its speed is increased.   Polymers based on TRIM, a trifunctional crosslinker, are a series of di- and tri- For peptides, resolution and capacity are higher than for EDMA-based polymers. It has already been reported that it is much better. The suspension polymerization method according to the present invention To evaluate Boc-L-Phe imprints on TRIM based polymers Manufactured in. Beads produced using 100 mg of PFPS (PF15) Beads with a diameter of 5.7 μm and made with 25 mg of PFPS (PF16) have an average 18.8 μm in diameter, which uses the same amount of stabilizing polymer and bases EDMA This is very close to the value expected to be obtained for the polymer used. in this way, With respect to bead size prediction, TRIM and EDMA have very similar properties. It is thought to show. An SEM image of the PF15 beads is shown in FIG. 2e. These When tested by HPLC, the size of the block Listed Such an excellent division was possible. This packed column has very low back pressure, Advanced separation could be achieved even at very high flow rates. Figures 4a-4e show that the flow rate is 0.5-5 ml / min, i.e., 0.5 ml / min, 1 ml / min, 2 ml / min, 3 ml / min and 5 ml / min. It shows the chromatogram of the run. Between 0.5 ml / min and 2 ml / min Almost no difference was observed, indicating that the diffusion rate of these small beads was high. It suggests. Back pressure was very low and resolution was excellent (0.5, 1 and 2 ml F / g = 0.89, 0.89, and 0.85, respectively). 5 ml / min (back pressure 91. 4 kg / cm^Two, 1300 psi) provided reasonable resolution (f / g = 0.61). Crush Random particles smaller than 25 μm are usually screened at flow rates above 1 ml / min. Did not give enough results. Using 5 μm crushed bulk polymer Difficult to work with. How to avoid high back pressure in HPLC columns Although various restrictions are required, sieving less than 10 μm is not possible, An alternative size sorting method is required. The result is Polymer can be used to prepare the column, as well as the performance of the resulting column. Has significant advantages over "traditional" ground and sieved block polymers. Suggests that   In addition, the stabilizer of the present invention is basically chemically inert, and thus is a water-sensitive monomer. -Units, for example for the production of granular polymers containing acid chlorides or anhydrides, It is believed that it can be used as a dispersant.   The foregoing invention has been described in detail, using figures and examples, to facilitate understanding thereof. However, the skilled artisan is aware of the requirements described herein and defined in the appended claims. Some changes may be made without departing from the spirit and scope of the invention as described. It is clear that improvements and improvements are possible.

[Procedure amendment] [Submission date] January 23, 1998 [Correction contents]                              Full text correction statement          Molecularly imprinted granular polymers and their polymers              Stabilized suspension polymerization in perfluorocarbon liquidField of the invention   The present invention relates to a molecularly imprinted polymer support. The present invention relates to a carrier and a method for producing these supports. More specifically, the present invention provides the following formula: Using a suspension polymerization method using a stabilized copolymer having olecule) around the polymerizable functional monomer. Where:   X is C_nF_{2n + 1}, C_nF_{2n + 1}(CH_Two)_rO-, C_nF_{2n + 1}O- or C_nF_{2n + 1}SO_Two N (C_TwoH_Five) C_TwoH_Four-O;   Y is (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four-O or (Z)_tCH_ThreeO (CH_TwoCH_Two O)_m-Where Z is a printed molecule;   n is from 1 to 20;   m may be from 0 to about 500;   p is at least 1;   q may be 0 or a positive number;   r is 1-20, preferably 1 or 2;   s is 0 or 1; and   t is 0 or 1. The polymeric support of the present invention is preferably in a granular form, which comprises amino acids, amino acid derivatives. Enables the separation or resolution of the body, pharmaceutical compounds and polysaccharides.Background of the Invention   Molecular imprinting, also called templating, Is used for chiral separation, which includes a polymerizable functionality around the print molecule. And arranging the functional monomers. This is the print molecule and the functional monomer When Using non-covalent interactions such as hydrogen bonding, ion pair interactions, etc. (Non-shared imprint) or by its reversible covalent interaction (shared imprint) Print). The resulting complex is further combined by polymerization to form a highly crosslinked porous It is a polymer matrix. When the printed molecules are extracted, special A site with an irregular shape and functional groups corresponding to the original print molecule remain. Mosbach, K., Tr ends, Biochemical Sciences,7, 92-96 (1994); Wulff, G., Trends, Biote. chnology,1185-87 (1993); and Andersson et al., Bioseparations (Ngo. Ed.), Pp. 383-394, Molecular Interactions.   Various racemates are resolved by molecular imprinting. And ersson et al., Bioseparations (Ngo.TT), Plenum Press, pp. 383-394 (19). 93) "Amino acid derivatives" in Molecular Interactions; .Am.Chem.Soc.,113, 9358-9360 (1991); "Drugs"; Kempe et al., J. C. hromatogr. 、664, 276-279 (1994); Wulff et al., J. Org. Chem.,56, 395-400 (1 "Sugars" in 991); and Mayes et al., Anal. Biochem.,222, 483-488 (1994) See In many cases, Baseline resolution is performed.   In contrast to other chiral stationary phases, the benefits of molecularly imprinted polymers The point is that the elution order of the enantiomers is predictable. Imprintable support The support is manufactured by a bulk polymerization method, in which a block of porous polymer is used. For the production, a porogenic solvent is used. But bulk The polymeric support is crushed and ground when used in separation columns and analytical protocols, It must be sieved to an appropriate particle size. For example, chromatography In the case of the measurement using a polymer, polymer particles of less than 25 μm are usually used. But bal In order to supply such small particles in the polymerization method, a pulverization method must be used. , This is not satisfactory. Grinding methods involve irregularly shaped particles and excess Undesirable amounts of "fines" are obtained. Usable among normally ground polymers Less than 50 percent (50%) is recovered as functional particles. Generally irregular Column cannot be efficiently packed with small particles and scale-up It has been found that difficulties are often encountered during the backup stage. But well-formed grains Particles, such as particulate polymers, may be preferred in most cases. Also powder Crushing method Requires additional processing steps to remove fines, i.e., settling steps. I do. This is expensive and time consuming. Bulk polymerization and the necessary grinding The law makes the effort of this conventional law severe, wasteful and unacceptable.   Manufacture beads from acrylic monomers that can contain imprinted molecules Therefore, attempts have been made to use suspension and dispersion polymerization methods. In theory, this These suspension and dispersion polymerization methods could be an alternative to bulk polymerization. You. However, existing suspension and dispersion methods use relatively hydrophobic monomers. Water or a highly polar organic solvent (eg, alcohol) as the continuous phase It is not satisfactory because of its use. These solvents are Has a specific interaction with the solvent, and is likely to compete with the functional monomer for the solvent. It is incompatible with most covalent and noncovalent imprint mixtures. In the suspension polymerization method , Because the solvent is used in a considerable molar excess, the solvent saturates the monomer phase and Significantly reduce the number and strength of interactions between the reactive monomers and the print molecules. Further In addition, acidic monomers are highly soluble in water, and may No coalescence or crosslinking occurs. Water-soluble print molecules are distributed in the aqueous phase. It is lost to spill. Not unexpected, but suspension polymerization in water Attempts to produce molecularly imprinted polymer beads Attempts have yielded very poor results. Damen et al., J. Am. Chem. Soc.,102, 3265-3267 (1980); Braun et al., Chemiker-Zeitung,108, 255-257 (1984) There is a stable covalent or metal chelate bond between the functional monomer and the print molecule. In some cases, use in aqueous conditions will be possible.   Also imprinted in the pore network of the finished granular silica To produce composite beaded particles Attempts have been made. Norrlow et al., J. Chromatogr.,299, 29-41 (1984); Wul. ff, Reactive Polymers,Three, 261-2757 (1985) or TRIM. But This method requires careful operation and requires additional The amount of imprinted polymer is necessarily reduced by the beads themselves.   Sellergren, B., J. Chromatogr.,673, 133-141 (1994) and Sellergren, B., Anal. Chem.,66, 1578-1582 (1994) states that polar solvents are mixed in molecular imprints. The use of dispersion polymerization in compounds has been reported. This method uses a regular bead A random sediment is obtained rather than a sediment. Evaluable results are highly This is probably due to the fact that charged molecules were obtained. Probably due to competing solvent effects.   Therefore, it is simple, reproducible and without compromising the quality of the resulting imprint Granular poly, including molecular imprints that do not require additional grinding and sieving equipment There is a need for Ma's recipe. The molecularly imprinted polymer is uniform Something is also required.Summary of the Invention   The present invention relates to a molecularly imprinted polymer support, and to that by suspension polymerization. Related to their manufacture. The suspension method according to the present invention employs polyoxyethylene Molecular imprinting method using perfluorocarbon liquid containing steal group I will provide a. Compounds containing perfluorocarbon-polyoxyethylene esters Compounds are required for the recognition process during molecular imprinting. It does not interfere with the interaction between the functional monomer and the print molecule. "Support" grains By changing the amount of stabilizing polymer or the method of stirring, the size of the Adjustments from μm to about 100 μm are possible.   It is therefore an object of the present invention to use imprinted polymers in granular and It is an object of the present invention to provide a method which can be easily produced in almost quantitative yield of possible substances.   Another object of the present invention is to provide a functional monomer and a printed component during suspension polymerization. Fluorocarbons that stabilize emulsions without disturbing interaction with the To provide a copolymer.   Further objectives include functional monomers, crosslinkers, print molecules, initiators and porologies. To stabilize the emulsion of the enic solvent.   It is another object of the present invention to provide a molecularly in size of about 2 μm to about 100 μm. It is to provide printed polymer beads in high yield.   It is a further object of the present invention to provide an excellent separation at low back pressure, high speed diffusion and higher flow rates. The aim is to provide a small bead packing of about 2-5 μm that results in separation.   These and other objects and advantages will be more fully understood from the following description and accompanying figures. Will be clear.Brief description of figures   FIG. 1 shows 1.84 g of EDMA, 0.16 g of MAA, 4.2 g of chromatogram in 20 ml of PMC. Added for "standard polymerization" containing emulsified formol and 20 mg of AIBN FIG. 4 is a graph showing the diameter of beads with respect to the amount of PFPS.   Figures 2a-2e show beads produced by suspension polymerization in PMC according to the invention. 3 is a scanning electron micrograph of the sample. Here the beads are placed on aluminum pegs And use a Polaron E5150 coater to spatter it with 15 mm gold. I did it. Images were taken at 25 kV using an ISI 100A SEM. Magnification is 500x It is. 2 (a) PF2; 2 (b) PF10; 2 (c) PF13; 2 (d) PF14; 2 (e) PF15.   3a-3e show a 25 cm column of Example PF15 (5 μm TRIM beads). It shows HPLC traces when Boc-D and L-Phe were separated. Condition: mosquito The rum was equilibrated with chloroform + 0.25% acetic acid; 1 mg in 20 μl mobile phase Of Boc-D, L-Phe, and using the same solvent, the flow rate was 3 (a) 0.5 ml / min; 3 (b) 1 ml / Min; 3 (c) 2 ml / min; 3 (d) 3 ml / min; 3 (e) 5 ml / min. 0.5 ml / min , F / g = 0.89, Rs = 1.36 and α = 1.52, the f / g values were 0.5, 1, 2, 3 and 5 ml At / min, they were 0.89, 0.89, 0.85, 0.76 and 0.61, respectively.Detailed description of the invention   According to the present invention, a liquid perfluorocarbon containing a polyoxyethylene ester group Based on an emulsion of a non-shared imprint mixture formed in the bon A suspension polymerization method is provided. Most of the conventional suspension and dispersion methods are relatively sparse. Water or a highly polar organic solvent (e.g., alcohol) as the mobile phase for aqueous monomers Call), which is not satisfactory in this regard. Therefore, another An approach is needed. The present invention provides this "alternative approach". It is. In addition, there are problems with conventional methods, such as specific interactions with printed molecules. Mostly shared and non-shared due to competition between solvent and functional monomer Solvents that are incompatible with the imprint mixture are avoided. In the present invention, print Use dispersants to prevent the interaction required for recognition between the molecule and the functional monomer. You do not need to know. In the present invention, a monomer, a crosslinking agent, a print molecule, a porogenic solution Liquid particles containing a solvent and a fluorinated surfactant. perfluorocarbons including polyoxyethylene ester groups Use a polymer. At this time, the number of print molecules for imprinting the surface is one. Sometimes it's a cord, sometimes it's not.   The stabilizer / dispersant of the present invention generally has the formula: Where:   X is C_nF_{2n + 1}, C_nF_{2n + 1}(CH_Two)_rO-, C_nF_{2n + 1}O- or C_nF_{2n + 1}SO_Two N (C_TwoH_Five) C_TwoH_Four-O;   Y is (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four-O or (Z)_tCH_ThreeO (CH_TwoCH_Two O)_m-Where Z is a printed molecule;   n is from 1 to 20;   m may be from 0 to about 500;   p is at least 1;   q may be 0 or a positive number;   r is 1-20, preferably 1 or 2;   s is 0 or 1; and   t is 0 or 1. C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Four-OH and (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four Blocks, triblocks, and mals formed from acrylated monomers of -OH Thiblock copolymers are also included in the scope of the present invention. Preferred Stability / Dispersion of the Invention The agent has the formula:             C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_FourO-CO-CH = CH_Two And a monomer A defined by the formula:             CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_FourO-CO-CH = CH_Two Wherein n is from 1 to 20 and m is 0 to 500. Preferably, n is 7.5 and m is about 43. Most preferred A new stabilizer / dispersant has the formula: This means that spherical beads can be produced in high yield by the suspension polymerization method. it can. Where:   X is C_nF_{2n + I}SO_TwoN (C_TwoH_Five) C_TwoH_Four-O;   Y is Z_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_FourAt -O, Z is a printed molecule;   n is from 1 to 20;   m may be from 0 to about 500;   p is at least 1;   q may be 0 or a positive number;   s is 0 or 1; and   t is 0 or 1. Preferably, m = about 7.5, n = about 43, p = about 19, and q = about 1. m, n, The values of p and q need not necessarily be integers.   By using the stabilizer of the present invention, the printing molecule and the functional monomer Using non-covalent interactions such as hydrogen bonding, ion-pair interactions, etc. Imprint) or by reversible covalent interaction (shared imprint) The purpose of the physical properties of the dose is achieved. The formed complex is further added by polymerization. Highly crosslinked porous polymer obtained from copolymerization of different acrylic monomers Mermatrix. When the printed molecules are extracted, special The site of the shape and the functional groups corresponding to the original print molecule remain.   Print molecules that can be used in the present invention include, but are not limited to: It is not something to be done.   1. D- and L-Boc tryptophan   2. D- and L-Boc phenylalanine   3. D- and L-phenylalanine   4. D- and L-Boc-proline-N-hydroxysuccinimide ester   5. D- and L-Cbz-tryptophan   6. D- and L-Cbz-aspartic acid   7. D- and L-Cbz-glutamic acid   8. D- and L-Cbz-tryptophan methyl ester   9. Nitrophenyl α and β, D-, L-galactoside   Ten. (S)-(-)-Timolol   11． D-fructose   12． D-galactose   13. Phenyl α-D-mannopyranoside   14. Acrylic α and β-glucoside   15． (R) -phenylsuccinic acid   16． Ac-L-Trp-OEz   17． L-PheβNA   18． L-LeuAn   19. L-PheAn   20. L-PheGlyAn   twenty one. L-MenPheAn   twenty two. L-PyMePheAn   twenty three. L-PLPheAn   twenty four. N-Ac-L-Phe-L-Trp-OMe   twenty five. Diazepam   26. Propranolol   27. ephedrine   However, in the following non-limiting examples, Boc-D- and L-Phe, and L-Phe were used. Was.Preparation of reagents monomer   Ethylene glycol dimethacrylate (EDMA) and methacrylic acid (MAA) (Merck, Darnstadt, Germany) was vacuum distilled before use. Trimethacrylic acid Limethylolpropane (TRIM) (Aldrich Chemie, Steinheim, Germany) , Styrene (Aldrich), methyl methacrylate (MMA) (Aldrich) and meth Benzyl acrylate (BMA) (Polysciences, Warringt, Mass.) on) was used as is. 2,2'-azobis (2-methylpropionitrile) (A IBN) was obtained from Janssen Chimica, Goel, Belgium.   2- (N-ethylperfluoroalkylsulfonamide) ethanol (PFA-1 ) (Fluorochem, Old Glossop, UK), PEG2000 monomethyl ether (MME) (Fluka Chemical AG, Buchs, Switzerland) and PEG 350 MME ( (Sigma, St. Louis, Missouri) is a trademark of Acryloyl Chloride in dichloromethane. And acrylates by reaction with triethylamine. Purchase is also possible (from Fluorochem and Polysciences, respectively). Perful Oro (methylcyclohexane) (PMC) and Fluorad FC430 are also Obtained from Fluorochem.Imprint molecule   Boc-D-Phe, Boc-L-Phe and Boc-D, L-Phe were purchased from Bachem AG. (S (Boc = t-butoxycarbonyl; Phe = Fe) Nylalanine).Porogenic solvent   Chloroform (CHCl_Three) (For HPLC) Ethanol is removed through a basic alumina column to use as a solvent Then, molecular sieves were put and stored. Used directly for HPLC. G Ruene was sodium and acetone was dried over molecular sieves before use. other The solvent of the grade for analysis or higher was used as it was.Preparation of stabilizer   The emulsion stable / dispersed polymers (PFPS) according to the invention are mostly Which formula: Where:   X is C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Four-O;   Y is Z_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_FourAt -O, Z is a printed molecule;   n is from 1 to 20;   m may be from 0 to about 500;   p is at least 1;   q may be 0 or a positive number; and   t is 0 or 1; In particular, n is about 7.5, m is about 43, p is about 19, and q is about 1.Example 1   4 g (7.2 mmol) of acryloyl PFA-1             C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Four-O-CO-CH = CH_Two And 0.76 g (0.36 mmol) of the formula:             CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four-O-CO-CH = CH_Two Dissolve acryloyl PEG2000 MME as defined in 10 ml of chloroform Was. In the above formula, n is about 7.5 and m is about 43. 24 mg (76 μmol) of AIBN And dissolved oxygen was removed by flowing nitrogen for 5 minutes. And this tube Was sealed and the contents polymerized in a shaking water bath at 60 ° C. for 48 hours. The resulting solution Was slightly turbid, but became more turbid when cooled. Solvent slowly at 30 ° C under reduced pressure It is almost distilled off at 60 ° C under reduced pressure to avoid foaming. Removed. The resulting polymer is a sticky, pale yellow paste, which is It was used without any further treatment.   Other polymer stabilizers use monomers and 1 mole% AIBN in appropriate ratios, Synthesized in a similar manner. All is obtained from cream as an amber polymer, which They varied from glassy to soft paste depending on their composition.Suspension polymerization   Determine the amount of porogenic solvent needed to just saturate 20 ml of PMC. Specified. In a 50 ml borosilicate glass tube, add the required amount of PFPS to the same amount of Porogeni. Solution, and add 20 ml of PMC to it to make a milky emulsion. , Shake until uniform white. 5 ml of “imprint mixture” (Table 1) In addition, the mixture was emulsified while stirring at 2000 rpm for 5 minutes.   * Beads made according to the present invention are listed in the first column of the table under the PF code number. This is the same in the comparative example.   The polymerization equipment used in all polymerizations of the present invention contained 50 ml of borosilicate glass. This tube has a screw cap and a stainless steel tube in the center. A hole has been drilled through the rotating shaft of a stainless steel flat blade stirrer. You. This flat blade stirrer is about 1.5 times the length of the tube. Evaporation through this hole A rubber seal was used to control. There is another small hole in the lid It can be used to send a stream of nitrogen into the tube. Stand retort tube Stand vertically in a (retort stand) and stir with an overhead stirrer. About 5 from tube  Place the UV lamp at a distance of cm and combine it with the lamp to maximize reflected light. The tube was wrapped in aluminum foil.   Prepare an emulsion of PF1-PF16 in Table 1 above and pass it under nitrogen for 5 minutes. The emulsion is then placed in a cooled nitrogen atmosphere at room temperature. While stirring at a flow rate of 500 rpm, UV of 366 nm was irradiated to perform polymerization. Polymerization is 3 Continued for hours. The obtained polymer particles (beads) are filtered with a glass filter, and PMC Was recovered. Wash the beads thoroughly with acetone to remove loose particles After sonication and crushing (large lumps are gently crushed and crushed with a spatula) It was dried and stored.   Adjust the size of beads by changing the amount of PFPS used during polymerization FIG. 1 shows this relationship. FIG. 1 illustrates the use of different amounts of PFPS in accordance with the present invention. 5 is a graph of the average value and the standard deviation of the beads manufactured as described above. Standard is 5 ml total volume Prepared using 2 g of monomer in it (see Table 1). Form a stable emulsion The minimum amount of PFPS possible is 10 mg, but this sample is also very agglomerated. I was in it. When PFPS is used at 150 mg or more, extremely small and irregular particles of 1-2 μm Only a child was obtained. No beads were found in these samples.Comparative example   Two commercially available fluorinated surfactants (polyfluoroalcohol (PFA-1) and Luorad 430), homopolymer of acryloyl PFA-1, acryloyl PFA-1 Series of random copolymers of styrene and styrene, methyl methacrylate or methacryl Benzyl acrylate and acrylates with perfluoroester and PEG groups Graft copolymers containing the main chain were also evaluated as stabilizers. However, this Most of them turned out to be ineffective. Emulsions quickly “creamy The reaction is hindered by the high density of dispersant generated, The dispersed droplets coalesced.   When polyacryloyl PFA-1 was evaluated alone, it was found that To give emulsions with sufficient stability to give good quality beads. Unfortunately Of the beads formed on the extremely hydrophobic surface after polymerization due to their low solubility. It was very difficult to remove this surfactant from the surface.   Copolymer of acryloyl-PFA-1 and acryloyl-PEG2000MME ( The most effective emulsions can be obtained using a molar ratio of 20: 1 with the latter being PFPS). Was.Properties of molecularly imprinted beads size   The type of emulsification depends on the polymerization method and the resulting molecularly imprinted beads. Affect. A preferred method involves stirring at about 2000 rpms for about 5 minutes. With this method, beads with a size of 2 to 25 μm have excellent uniformity. And reproducibility. Five separate polymerizations using 25 mg of PFPS as emulsifier The average bead size was 19.7 μm and the standard error was 0.6 μm.   Emulsification in an ultrasonic bath for 5 minutes, including significant amounts of small particles, Distribution was very extensive. If you have larger beads, i.e. 40 μm If you want 100 μm beads, shake well 3-4 times normally in a tube. The fruit was obtained.   The polymerization temperature also affected the polymerization method and the resulting beads. ABDV as initiator Tried to start with heat at 45 ° C, but only small and irregular pieces were obtained. Was done. Initiating polymerization by UV at 4 ° C resulted in very large masses wait. Due to the proximity to the UV lamp, the temperature increased somewhat during the polymerization, Most polymerizations were performed at ambient temperature (about 20 ° C), ie, above about 18 ° C. very In a warm climate, that is, when the ambient temperature reaches 30 ° C, Small beads are obtained, which gives reproducible results , It has been suggested that the polymerization temperature can also affect the size of the beads.   Further, the polymerization is carried out in a range of solvents commonly used in molecular imprinting, for example, Runs were also made in lume, toluene, acetonitrile and acetone. In this polymerization method, All of these solvents can be used and are therefore almost always used for imprinting. Therefore, this law is appropriate. However, preferred solvents are chloroform and toluene. And acetone. The size and surface structure of the obtained beads are It also depends on the nick solvent. In a "standard" polymerization, 25 mg PFPS was used. If toluene (26 μm ± 12 μm-mean ± SD) is also acetone (52 μm ± 15 μm) Also gave larger beads than chloroform (18 pm ± 8 pm).Bead size distribution   The beads were suspended in acetone, dried on a microscope slide, and Approximately 150 beads were measured randomly using the reference line of the microscope. The magnification is Either 100 × or 400 × was used depending on the particle size. Some sumps Le also filmed with SEM. The measurements obtained from these images are optical measurements Well agreed with the results obtained fromSEM   Place polymer beads on aluminum pegs, Polaron E5150 Gold Sputter coating was performed with 15 nm gold using a coater. Furthermore, under different conditions To compare the size, surface and pore structure of the manufactured beads, ISI 100A Images were obtained at 25 kV using SEM. Scanning electrons of some granular polymer products Micrographs are shown in FIGS. 2a-2e. The method of the present invention can be used for various porogenic solutions. By using a medium, substantially spherical beads, that is, EDMA (2a-2 d) and give a polymer based on TRIM (2e). Surface depression The incidence of defects such as small holes is due to water-based suspension polymerization in water. Somewhat higher than the incidence normally observed in The form of the beads is suitable for suspension polymerization. Thus, as symbolized by the beads made, they are slightly denser and more slippery. The kana surface covers the more porous structure inside.   Beads made using acetone as a porogenic solvent (FIG. 2d) Was different. They are manufactured using chloroform or toluene Larger and the surface morphology is very rough, and moreover, There were many "debris". Using toluene as a porogenic solvent The beads produced have a lower surface shell density than those produced with chloroform. The internal structure was somewhat porous. FIG. 2b shows beads of polymer PF9. However, this has a lower percentage of crosslinking than the beads (polymer PF2) of FIG. 2a. The beads in FIG. 2b are much more irregular and distorted, indicating that their particles Are still much softer and more prone to deformation, so It suggests that it tends to be distorted. The internal form of these beads is a polymer It is also clear that it is more open and porous than PF2 beads. this This may contribute to the latter higher HPLC performance. FIG. The beads in (c) are PF13 beads, and the beads in FIG. 2 (e) are PF15 beads. Is shown.HPLC   The polymer beads produced according to the invention are molecularly imprinted And the properties of the recognition sites are at least as high as those obtained by conventional bulk polymerization. Imprint using Boc-Phe to confirm that it is as good as A series of polymers were evaluated by HPLC. This system is available at Boc-Phe There is a great deal of information on the properties of traditionally milled bulk polymers that have been imprinted. It was chosen because it can be obtained.   Suspend beads in chloroform-acetone mixture (17: 3) by sonication And slurries the slurry into 10 cm x 0.46 cm or 25 cm x 0.46 cm stainless steel. In a column, use an air driven fluid pump to remove the solvent. Filled at 300 bar as seton. Use 250 ml of methanol: vinegar Wash with acid (9: 1) and wash with chloroform containing 0.1% or 0.25% acetic acid. I was in equilibrium. 10 μg Boc-D or L-Phe in 20 μl solvent, or 20 μg And the chromatogram was recorded at 254 nm at a flow rate of 0.5 ml / min. . Also, some separations use higher flow rates and more loading compounds. I went. Chromatographic parameters were calculated using standard theory .   The results for the HPLC evaluation of the six polymers are summarized in Table 2. As the ratio of MAA to print molecule increases, non-specific interactions increase. Retention time is increased, which further increases the capacity factor. ors) increase. However, the α value is almost constant at about 1.8. This value is The values obtained under the same conditions for the crushed and sieved bulk Boc-Phe polymer (1. (Range 77 to 2.17). The separation was performed with a MAA: Boc-Phe ratio of 12: 1.  It was found that the best value was 0.83 for the 10 cm column. Polymer -Using a longer column of PF11 (25 cm), bulk polymer (chromatography) Rum is not shown)). The result was close to the splitting. Polymer PF9 with only 62.5% cross-linking G: MAA ratio is the same, but cross-linking is better than 75% polymer PF11 The result was obtained. Previously, it was shown that increasing the degree of cross-linking within this range improves separation. However, this was not observed in these examples. These It is impossible to change the parameters independently, increasing the number of binding sites, Whether the separation improves even if the polymer morphology is changed by reducing cross-linking PF11 was manufactured by reducing the amount of print molecules used. (See Table 1). From these observations, it can be seen that the improvement in separation depends on many compositions and operations. It can be seen that this can be achieved by carefully optimizing the variables. Bee It can be even better if the polymerization method is simplified and its speed is increased.   Polymers based on TRIM, a trifunctional crosslinker, are a series of di- and tri- For peptides, resolution and capacity are higher than for EDMA-based polymers. It has already been reported that it is much better. The suspension polymerization method according to the present invention To evaluate Boc-L-Phe imprints on TRIM based polymers Manufactured in. Beads produced using 100 mg of PFPS (PF15) Beads with a diameter of 5.7 μm and made with 25 mg of PFPS (PF16) have an average 18.8 μm in diameter, which uses the same amount of stabilizing polymer and bases EDMA This is very close to the value expected to be obtained for the polymer used. in this way, With respect to bead size prediction, TRIM and EDMA have very similar properties. It is thought to show. An SEM image of the PF15 beads is shown in FIG. 2e. These When tested by HPLC, the size of the block Listed Such an excellent division was possible. This packed column has very low back pressure, Advanced separation could be achieved even at very high flow rates. Figures 3a-3e show that the flow rate is 0.5-5 ml / min, i.e. 0.5 ml / min, 1 ml / min, 2 ml / min, 3 ml / min and 5 ml / min It shows the chromatogram of the run. Between 0.5 ml / min and 2 ml / min Almost no difference was observed, indicating that the diffusion rate of these small beads was high. It suggests. Back pressure was very low and resolution was excellent (0.5, 1 and 2 ml F / g = 0.89, 0.89, and 0.85, respectively). 5 ml / min (back pressure 91. 4 kg / cm^Two, 1300 psi) provided reasonable resolution (f / g = 0.61). Crush Random particles smaller than 25 μm are usually screened at flow rates above 1 ml / min. Did not give enough results. Using 5 μm crushed bulk polymer Difficult to work with. How to avoid high back pressure in HPLC columns Although various restrictions are required, sieving less than 10 μm is not possible, An alternative size sorting method is required. The result is Polymer can be used to prepare the column, as well as the performance of the resulting column. Has significant advantages over "traditional" ground and sieved block polymers. Suggests that   In addition, the stabilizer of the present invention is basically chemically inert, and thus is a water-sensitive monomer. -Units, for example for the production of granular polymers containing acid chlorides or anhydrides, It is believed that it can be used as a dispersant.   The foregoing invention has been described in detail, using figures and examples, to facilitate understanding thereof. However, the skilled artisan is aware of the requirements described herein and defined in the appended claims. Some changes may be made without departing from the spirit and scope of the invention as described. It is clear that improvements and improvements are possible.                                The scope of the claims   1. formula:             C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_FourO-CO-CH = CH_Two And a monomer unit A defined by:             CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_FourO-CO-CH = CH_Two A copolymer containing a monomer unit B defined by the formula: wherein n is from 1 to 20. A suspension polymerization stabilizer wherein m can be from 0 to about 500.   2. The stabilizer of claim 1, wherein n is about 7.5 and m is about 43.   3. formula: Where:       X is C_nF_{2n + 1}, C_nF_{2n + 1}(CH_Two)_rO-, C_nF_{2n + 1}O- or C_nF_{2n + 1} SO_TwoN (C_TwoH_Five) C_TwoH_Four-O;       Y is (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four-O or (Z)_tCH_ThreeO (CH_TwoC H_TwoO)_m-Where Z is a printed molecule;       n is from 1 to 20;       m may be from 0 to about 500;       p is at least 1;       q may be 0 or a positive number;       r is 1-20, preferably 1 or 2;       s is 0 or 1; and       A polymer in which t is 0 or 1.   4. formula: Where:       X is C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Four-O;       Y is CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four-O;       n is from 1 to 20;       m is from 0 to about 500;       p is at least 1; and       q is a polymer which is 0 or a positive number.   5. In liquid perfluorocarbons containing polyoxyethylene ester groups And stable emulsions including emulsions in which imprint molecules are emulsified.   6. The emulsion of claim 5 wherein said liquid fluorocarbon is a copolymer. Yeah.   7. The copolymer has the formula:             C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Four-O-CO-CH = CH_Two And a monomer A as defined by             CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four-O-CO-CH = CH_Two Wherein n is from 1 to 20 and m is 7. The emulsion of claim 6, wherein is from 0 to about 500.   8. Imprint molecule and formula: Where:       X is C_nF_{2n + 1}, C_nF_{2n + 1}(CH_Two)_rO-, C_nF_{2n + 1}O- or C_nF_{2n + 1} SO_TwoN (C_TwoH_Five) C_TwoH_Four-O-;       Y is (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four-O- or (Z)_tCH_ThreeO (CH_Two CH_TwoO)_m-Where Z is a printed molecule;       n is from 1 to 20;       m may be from 0 to about 500;       p is at least 1;       q may be 0 or a positive number;       r is 1-20, preferably 1 or 2;       s is 0 or 1; and       An emulsion containing a polymer in which t is 0 or 1.   9. Imprint molecule and formula: Where:       X is C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Four-O;       Y is CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four-O;       n is from 1 to 20;       m is from 0 to about 500;       p is at least 1; and       An emulsion containing a polymer in which q is 0 or a positive number.   10. a) 1) a mixture of different acrylic monomers;           2) at least one crosslinking agent;           3) at least one initiator;           4) at least one molecularly imprinted molecule; and           5) at least one porogenic solvent           Forming a reaction mixture comprising: and       b) Perflu to form molecularly imprinted polymer particles In the presence of a stabilizer containing orocarbon and polyoxyethylene ester groups, A suspension polymerization method comprising polymerizing the reaction mixture.   11. The method according to claim 10, wherein the stabilizer is chemically inert.   12. The acrylic monomer is methacrylic acid and ethylene glycol dimethacrylate. The method of claim 10, wherein the call is a call.   13. The stabilizer has the formula: A polymer defined by the formula:       X is C_nF_{2n + 1}, C_nF_{2n + 1}(CH_Two)_rO-, C_nF_{2n + 1}O- or C_nF_{2n + 1} SO_TwoN (C_TwoH_Five) C_TwoH_Four-O-;       Y is (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four-O- or (Z)_tCH_ThreeO (CH_Two CH_TwoO)_m-Where Z is a printed molecule;       n is from 1 to 20;       m may be from 0 to about 500;       p is at least 1;       q may be 0 or a positive number;       r is 1-20, preferably 1 or 2;       s is 0 or 1; and       The method according to claim 10, wherein t is 0 or 1.   14． The stabilizer has the formula: A polymer defined by the formula:       X is C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Four-O;       Y is CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Four-O;       n is from 1 to 20;       m is from 0 to about 500;       p is at least 1; and       The method according to claim 10, wherein q is 0 or a positive number.   15. Before polymerization, an emulsion is formed from components 1-5 and the stabilizer. The method according to claim 10.   16. To form the emulsion, mix the reaction mixture at about 2000 rpms. 16. The method of claim 15, wherein mixing is performed for about 5 minutes.   17． The method according to claim 15, wherein the polymerization is carried out at a temperature of less than about 20 ° C to about 30 ° C.   18. The method according to claim 10, wherein the polymerization is initiated by irradiation with ultraviolet light.   19. The method according to claim 10, wherein the polymer particles are beads. Method.   20. The polymer particles provide a uniform surface for a reproducible surface presentation. 12. The method of claim 11, wherein the method is provided.   21. 17. The method of claim 16, wherein said beads are spherical.   22. 20. The method of claim 19, wherein said beads have a diameter of about 2 to 100 microns. Method.   23. 20. The method of claim 19, wherein said beads have a diameter of 2 to 50 microns. .   24. 20. The method of claim 19, wherein said beads have a diameter of about 5 to 25 microns. Law.   25. The porogenic solvent is more than acetone, chloroform and toluene. A method according to claim 10, which is selected.   26. The molecular imprinted molecule is an amino acid, an amino acid derivative, a pharmaceutical compound. The product according to claim 10, 13 or 14, wherein the substance and the polysaccharide can be separated or separated. The described method.   27. The molecularly imprinted molecules are D- and L-Boc amino acids, and 15. The method according to claim 10, 13 or 14, wherein the method is selected from derivatives of   28. Form beads with a uniform surface to present a reproducible surface Therefore, at least two different acrylic monomers and at least one Molecularly imprinted support formed from a printed molecule.   29. 29. The particle of claim 28, wherein said beads are spherical.   30. 30. The method of claim 29, wherein the beads have a diameter of about 2 to 100 microns. particle.   31. 31. The particle of claim 30, wherein said beads have a diameter of 2 to 50 microns. .   32. 32. The particle of claim 31, wherein said beads have a diameter of about 5 to 25 microns. Child.   33. The acrylic monomer is methacrylic acid and ethylene glycol dimethacrylate. 29. The particle of claim 28 which is Cole.   34. The molecular imprinted molecule is an amino acid, an amino acid derivative, a pharmaceutical compound. 29. Particles according to claim 28, capable of separating or resolving substances and polysaccharides.   35. The molecularly imprinted molecules are D- and L-Boc amino acids, and 35. The particle of claim 34 selected from derivatives of   36. The method according to claim 13 or 14, wherein s is 0.   37. 14. The method according to claim 13, wherein s is 1.   38. 14. The method of claim 13, wherein t is 1.   39. The printed molecule is D- and L-Boc tryptophan, D- and L-Boc Phenylalanine, D- and L-phenylalanine, D- and L-Boc-proline-N -Hydroxysuccinimide esters, D- and L-Cbz-tryptophan, D- and L-Cbz-aspartic acid, D- and L-Cbz-glutamic acid, D- and L-Cbz-tri Putphan methyl ester, nitrophenyl α and β, D-, L-galactoside, (S)-(-)-Timolol, D-fructose, D-galactose, phenyl α-D-ma Nonopyranoside, acrylic α and β-glucoside, (R) -phenylsuccinic acid, Ac-L -Trp-OEz, L-PheβNA, L-LeuAn, L-PheAn, L-PheGlyAn, L- MenPheAn, L-PyMePheAn, L-PLPheAn, N-Ac-L-Phe-L-Trp-O 2. A method according to claim 1, wherein said me is selected from Me, diazepam, propranolol and ephedrine. 3. The method according to 3. FIG. FIG. 2 FIG. 2 FIG. 2 FIG. 3

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI // C07B 57/00 310 C07B 57/00 310 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI , FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD) , TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ , BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, K Z, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ , TM, TR, TT, UA, UG, UZ, VN (72) Inventor Mosbash, Krauss S-244 02 Sweden Lölllund, Rakaranga 31 (72) Inventor Maze, Andrew G. Cambridgeshire, UK 19. 2 T.A., St. Neots, Ainsbury, St. Mary's Street 22

Claims (1)

[Claims]   1. formula:             C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_TwoO-CO-CH = CH_Two And a monomer unit A defined by:             CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_TwoO-CO-CH = CH_Two A copolymer containing a monomer unit B defined by the formula: wherein n is 1 A suspension polymerization stabilizer, wherein m is from 10 to 70 to about 500.   2. The stabilizer of claim 1 wherein n is about 7.5 and m is about 43.   3. formula: Where:       X is C_nF_{2n + 1}, C_nF_{2n + 1}(CH_Two)_rO-, C_nF_{2n + 1}O- or C_nF_{2n + 1} SO_TwoN (C_TwoH_Five) C_TwoH_Two-O;       Y is (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two-O or (Z)_tCH_ThreeO (CH_TwoC H_TwoO)_m-Where Z is a printed molecule;       n is from 1 to 20;       m may be from 0 to about 500;       p is at least 1;       q may be 0 or a positive number;       r is 1-20, preferably 1 or 2;       s is 0 or 1; and       A polymer in which t is 0 or 1.   4. formula: Where:       X is C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Two-O;       Y is CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two-O;       n is from 1 to 20;       m is from 0 to about 500;       p is at least 1; and       q is a polymer which is 0 or a positive number.   5. The stabilizer of claim 1 wherein n is about 7.5 and m is about 43.   6. In liquid perfluorocarbons containing polyoxyethylene ester groups And stable emulsions including emulsions in which imprint molecules are emulsified.   7. The emulsion of claim 6, wherein said liquid fluorocarbon is a copolymer. Yeah.   8. The copolymer has the formula:             C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Two-CO-CH = CH_Two And a monomer A as defined by             CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two-CO-CH = CH_Two Which is formed by the polymerization of monomer B as defined in The emulsion according to claim 7, wherein m is from 0 to about 500.   9. Imprint molecule and formula: Where:       X is C_nF_{2n + 1}, C_nF_{2n + 1}(CH_Two)_rO-, C_nF_{2 + 1}O- or C_nF_{2n + 1}S O_TwoN (C_TwoH_Five) C_TwoH_Two-O-;       Y is (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two-O- or (Z)_tCH_ThreeO (CH_Two CH_TwoO)_m-Where Z is a printed molecule;       n is from 1 to 20;       m may be from 0 to about 500;       p is at least 1:       q may be 0 or a positive number;       r is 1-20, preferably 1 or 2;       s is 0 or 1; and       An emulsion containing a polymer in which t is 0 or 1.   Ten. Imprint molecule and formula: Where:       X is C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Two-O;       Y is CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two-O;       n is from 1 to 20;       m is from 0 to about 500;       p is at least 1; and       An emulsion containing a polymer in which q is 0 or a positive number.   11． a) 1) a mixture of different acrylic monomers;           2) at least one crosslinking agent;           3) at least one initiator;           4) at least one molecularly imprinted molecule; and           5) at least one porogenic solvent           Forming a reaction mixture comprising: and       b) Perflu to form molecularly imprinted polymer particles In the presence of a stabilizer containing orocarbon and polyoxyethylene ester groups, A suspension polymerization method comprising polymerizing the reaction mixture.   12． The method according to claim 11, wherein the stabilizer is chemically inert.   13. The acrylic monomer is methacrylic acid and dimethacrylic acid ethylene glycol 12. The method of claim 11, wherein the method is   14. The stabilizer has the formula: A polymer defined by the formula:       X is C_nF_{2n + 1}, C_nF_{2n + 1}(CH_Two)_rO-, C_nF_{2n + 1}O- or C_nF_{2n + 1} SO_TwoN (C_TwoH_Five) C_TwoH_Two-O-;       Y is (Z)_tCH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two-O- or (Z)_tCH_ThreeO (CH_Two CH_TwoO)_m-Where Z is a printed molecule;       n is from 1 to 20;       m may be from 0 to about 500;       p is at least 1;       q may be 0 or a positive number;       r is 1-20, preferably 1 or 2;       s is 0 or 1; and       The method according to claim 11, wherein t is 0 or 1.   15． The stabilizer has the formula: A polymer defined by the formula:       X is C_nF_{2n + 1}SO_TwoN (C_TwoH_Five) C_TwoH_Two-O;       Y is CH_ThreeO (CH_TwoCH_TwoO)_mC_TwoH_Two-O;       n is from 1 to 20;       m is from 0 to about 500;       p is at least 1; and       The method according to claim 11, wherein q is 0 or a positive number.   16． Before polymerization, an emulsion is formed from component 1-5 and the stabilizer. The method of clause 11.   17． The reaction mixture is mixed at about 2000 rpms for about 5 to form the emulsion.  17. The method of claim 16, wherein mixing is performed for minutes.   18． 17. The method of claim 16, wherein the polymerization is performed at a temperature of less than about 20C to about 30C.   19. The method according to claim 11, wherein the polymerization is initiated by irradiation with ultraviolet light.   20. The method according to claim 11, wherein the polymer particles are beads. .   twenty one. The polymer particles provide a uniform surface for reproducible surface presentation 13. The method of claim 12, wherein the method comprises:   twenty two. The method according to claim 17, wherein the beads are spherical.   twenty three. 21. The method of claim 20, wherein said beads have a diameter of about 2 to 100 microns. Law.   twenty four. 21. The method of claim 20, wherein said beads have a diameter of 2 to 50 microns.   twenty five. 21. The method of claim 20, wherein said beads have a diameter of about 5 to 25 microns. .   26. The porogenic solvent is selected from acetone, chloroform and toluene. 12. The method of claim 11, wherein the method is selected.   27. The molecular imprinted molecule is an amino acid, an amino acid derivative, a pharmaceutical compound, And the polysaccharides can be separated or separated. the method of.   28. The molecularly imprinted molecule comprises D- and L-Boc amino acids, and their 16. The method according to claim 11, 14, or 15, wherein the method is selected from derivatives.   29. Form beads with a uniform surface to present a reproducible surface At least two different acrylic monomers and at least one imprint And molecularly imprinted support formed from   30. 30. The particles of claim 29, wherein said beads are spherical.   31. 31. The particle of claim 30, wherein said beads have a diameter of about 2 to 100 microns. Child.   32. 32. The particle of claim 31, wherein said beads have a diameter of 2 to 50 microns.   33. 33. The particle of claim 32, wherein said beads have a diameter of about 5 to 25 microns. .   34. The acrylic monomer is methacrylic acid and dimethacrylic acid ethylene glycol 30. The particle of claim 29 which is   35. The molecular imprinted molecule is an amino acid, an amino acid derivative, a pharmaceutical compound, 30. The particles of claim 29, wherein said particles are capable of separating or resolving polysaccharides.   36. The molecularly imprinted molecule comprises D- and L-Boc amino acids, and their The particle according to claim 35, selected from derivatives.   37. The method according to claim 14 or 15, wherein s is 0.   38. 15. The method according to claim 14, wherein s is 1.   39. 15. The method according to claim 14, wherein t is 1.   40. The printed molecules are D- and L-Boc tryptophan, D- and L-Boc Phenylalanine, D- and L-phenylalanine, D- and L-Boc-proline-N- Hydroxysuccinimide ester, D- and L-Cbz-tryptophan, D- and L -Cbz-aspartic acid, D- and L-Cbz-glutamic acid, D- and L-Cbz-tripp Tophan methyl ester, nitrophenyl α and β, D-, L-galactoside, ( S)-(-)-Timolol, D-fructose, D-galactose, phenyl α-D-man Nopyranoside, acrylic α and β-glucoside, (R) -phenylsuccinic acid, Ac-L- Trp-OEz, L-PheβNA, L-LeuAn, L-PheAn, L-PheGlyAn, LM enPheAn, L-PyMePheAn, L-PLPheAn, N-Ac-L-Phe-L-Trp-OM e, selected from diazepam, propranolol and ephedrine. The method described in.